Fruits are major components of the human diet contributing a large portion of vitamins, minerals, antioxidants, and fiber. While flavor and nutrition composition have clear and profound potential for positive human benefit, they have proven to be difficult traits to modify via either traditional breeding or transgenic approaches due to their generally complex biosynthetic and regulatory pathways. In fact, the biochemical descriptors that comprise flavor are poorly defined. What is typically perceived as flavor in many fruits is the product of a complex interaction among sugars, acids and multiple volatile secondary metabolites (Buttery et al., 1988; Baldwin et al., 2000). Synthesis and accumulation of these compounds is the result of coordinated activity of many genes that may also impact additional aspects of plant growth and development. Effective manipulation of these traits for human benefit will therefore require greater knowledge of the pathways involved and the regulatory systems which control them. Prior to the advent of genomics, researchers could focus on the activity of only one to several genes important in a process of interest and could view their respective effects in relative isolation. From a practical perspective, flavor and nutrition are intimately related and equally important as flavor directly impacts the choice of foods for consumption which, in turn, has positive nutritional consequences on the human diet.
Fruit-bearing crop plants are taxonomically diverse (e.g., pepper, tomato, melons, apples, bananas, grapes). However, they do share common features; most, though not all, fruits are enlarged ovaries. While our knowledge of how domesticated plants came to bear fruit or the mechanisms by which they ripen is still rudimentary, more is known about these processes in tomato (Lycopersicon esculentum) than in any other species (see Giovannoni (2001) for review). Furthermore, a diverse set of Near Isogenic Lines (NILs), single gene ripening mutants, and transgenic lines represent portals through which genetic regulation of fruit development and ripening can be studied (Gray et al., 1994; Giovannoni et al., 1999). The diversity of genetically well characterized tomato germplasm described below (Table 1) is unparalleled in other fruiting species. Nevertheless, it is important to realize that while fruit ripening is a complex sum of coordinately regulated biochemical events that vary from species to species, key regulatory components are likely to be maintained (Hobson and Grierson, 1993). For example, one group has recently identified two genes that are essential for fruit ripening, RIN and NOR (Giovannoni, 2001; Vrebalov et al., 2002). Fruit-specific, ripening-induced homologues of these genes have been identified from strawberry and banana (Vrebalov et al., 2002). Strawberry undergoes a very different ripening program as compared to tomato in that strawberry is non-climacteric (i.e. no increase in respiration or ethylene biosynthesis during ripening) and accumulates high levels of anthocyanins rather than carotenoids during fruit maturation. Further, it is anatomically a receptacle, whereas most fruits are ovaries. Banana is interesting in that while its fruit are also expanded carpels, it is a monocot. Apparently similar ripening control shared among monocots and dicots indicates that basic ripening regulation is likely conserved through evolution. In summary, these results suggest that while specific nutritional and flavor components may vary among fruit species they are likely due to regulated metabolic flux through similar pathways with similar genetic control systems. Thus, regulatory and biosynthetic genes identified in tomato will allow for modification of the same or related compounds in a wide range of agriculturally important fruit species.
Tomato has long served as a model system for plant genetics, development, physiology and fruit ripening resulting in the accumulation of substantial information regarding the biology of this economically important plant. Many experimental tools and features of tomato make it ideal for study of fruit ripening; these include extensive germplasm collections, numerous natural, induced, and transgenic mutants, routine transformation technology, a dense and expanding RFLP map, numerous cDNA and genomic libraries, a small genome, relatively short life-cycle and ease of growth and maintenance. In addition, numerous genomic tools that have and continue to be developed include: a) over 140,000 EST sequences (˜30,000 non-redundant) from 23 different tomato tissues/treatments (with one-third of the ESTs derived from fruit), b) EST expression arrays being developed and utilized (see bti.cornell.edu/CGEP/CGEP.html) and c) recent initiation of activities toward development of a tomato physical map anchored to the genetic map to facilitate gene isolation and eventual genome sequencing (Tanksley et al., NSF tomato genome project, 1992). The intense research effort in tomato fruit biology has resulted in many important discoveries that have had a broad impact on the field of plant biology, including control of gene expression by antisense technology, characterization of numerous genes influencing fruit development and ripening, characterization of genes for ethylene synthesis and perception, and the recent connection of ripening regulation and ethylene response to the molecular regulation of floral development (Vrebalov et al., 2002).
Fruit maturation and ripening is the summation of biochemical and physiological changes occurring at the terminal stage of development rendering the organ edible and valuable as an agricultural commodity. These changes frequently include modification of cell wall ultrastructure and texture, conversion of starch to sugars, alterations in pigment and nutrient biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Rhodes, 1980; Hobson and Grierson, 1993). While some ripening effects, such as carotenoid and vitamin C synthesis and accumulation, have direct impact on the nutritive value of mature fruit, others impacting flavor and texture (e.g., volatiles, sugars and acids) can have an indirect impact on human nutrition via their contributions to total consumption levels. In short, “if it tastes better” consumption will increase. This is especially critical as poor food choices exert a disproportional impact on children and members of society on lower rungs of the socio-economic ladder.
Although most fruits display modifications in color, texture, flavor and nutrient composition during maturation, two major classifications of ripening, climacteric and non-climacteric, have been utilized to distinguish fruit on the basis of respiration and ethylene synthesis rates. Climacteric fruits such as tomato, avocado, banana, peaches and apples, are distinguished from non-climacteric fruits such as strawberry, grape and citrus, by their increased respiration and ethylene synthesis rates during ripening (Lelievre et al., 1998). In tomato, ethylene has been shown to be necessary for the coordination and completion of ripening (Yang, 1985; Tucker and Brady, 1987; Klee et al., 1991; Picton et al., 1993; Lanahan et al., 1994). The critical role of ethylene in coordinating climacteric ripening at the molecular level was first observed via analysis of ethylene inducible ripening-related gene expression in tomato (Lincoln et al., 1987; Maunders et al., 1987). Numerous fruit development-related genes have since been isolated via differential expression patterns and biochemical function (reviewed in Gray et al., 1994). The in vivo functions of many fruit development- and ripening-related genes have been tested via antisense repression and/or mutant complementation in tomato. As examples, polygalacturonase was shown to be necessary for ripening-related pectin depolymerization and pathogen susceptibility, yet to have little effect on fruit softening (Smith et al., 1988, Giovannoni et al., 1989, Kramer et al., 1990). Inhibition of phytoene synthase resulted in reduced carotenoid biosynthesis and reduction in fruit and flower pigmentation (Fray and Grierson, 1993). Reduced ethylene evolution resulted in ripening inhibition of ACC synthase (ACS) and ACC oxidase (ACO) antisense lines (Oeller et al., 1991; Hamilton et al., 1990) while introduction of a dominant mutant allele of the NR ethylene receptor resulted in plants inhibited in virtually every measurable ethylene response including fruit ripening (Wilkinson et al., 1995; Yen et al., 1995).
Expression analysis of multiple tomato ripening-related genes indicates that a subset exhibit developmentally-controlled ethylene inducibility, i.e., they are ethylene inducible only in ripening fruits. Examples include members of the ACO and ACS gene families (Theologis et al., 1993; Blume and Grierson, 1997; Nakatsuka et al., 1998), the NR ethylene receptor (Wilkinson et al., 1995; Payton et al., 1996; Lashbrook et al., 1998) and E8 (Deikman et al., 1992). Additional evidence for non-ethylene mediated ripening control comes from analysis of gene expression in ripening impaired mutants such as rin (ripening-inhibitor) and nor (non-ripening) that fail to ripen in response to exogenous ethylene yet display signs of ethylene sensitivity and signaling including induction of some ethylene-regulated genes (Yen et al., 1995). These results suggest that regulatory constraints are placed on climacteric fruit maturation in addition to general ethylene biosynthesis and signaling. Such mechanisms could include fruit-specific regulation of certain subsets of ethylene regulated genes or factors that operate separate from and in addition to ethylene as seems to be the case for both the RIN (Vrebalov et al., 2002) and NOR transcription factors. This is particularly interesting as a greater understanding of the relationship between ethylene, developmental, and environmental signals will likely reveal the impact of various signaling systems on pathways impacting flavor and human nutrition. Indeed numerous environmental factors such as light and temperature can dramatically influence the degree and rate of fruit ripening with significant impacts on the accumulation of carotenoids and flavor compounds (Hobson and Grierson, 1993; Yen et al., 1997).
Numerous plant metabolites can be listed when the net of “nutritive compounds” is cast. These include various antioxidants, vitamins, minerals, fiber, lipids, and amino acids, to name just a few. In addition, as noted above, one can rationally argue that modification of flavor and additional quality attributes may lead to improved health via increased fruit or vegetable consumption.
Tomato fruits are among the highest source of lycopene, β-carotene, and vitamin C (ascorbate) in the diets of humans in the US, South America, and Europe, with steadily increasing prominence in Asia and the Middle East. In addition to direct nutritive value, carotenoids in particular are metabolized to compounds that impact flavor and aroma of fruit and thus have a significant impact on resulting fresh and processed products. Genes encoding the synthetic steps from phytoene through β-carotene (Bartley et al., 1994; Ronen et al., 1999) are potential regulatory points for modification of carotenoid levels. Indeed, available data indicate that accumulation of lycopene is due to coordinated up-regulation of the genes preceding its synthesis and down-regulation of genes that further metabolize it during ripening (Ronen et al., 1999). Numerous mutant, transgenic, RI and breeding lines that display a wide range of levels of lycopene and β-carotene are available (Table 1). While specific mutants represent some of the catalytic steps (e.g., r=phytoene synthase and cr and B=lycopene cyclase; Hamilton et al., 1990; Ronen et al., 1999) others such as hp-1 and hp-2 represent regulators of environmental response. Antisense phytoene synthase tomato lines are greatly reduced in all of the carotenoid-derived volatiles (Baldwin et al., 2000). Furthermore, transgenic and mutant lines altered in ethylene synthesis or perception display variation in carotenoid levels (Table 1).
TABLE 1Tomato germplasm altered in carotenoids, flavonoids, vitamin C.CarotenoidsVit. CVolatilesFunctionGenotyperin; ripening-inhibitor*very lowlowNAMADS-box proteinnor; non-ripening*lowlowNAtranscription factorNr: Never-ripe*lowNANAethylene receptorhp-2; high-pigment-2highhighNADET1 (light signaling)cr; crimsonlow B, high LhighNAlycopene cyclaseB; Betahigh B, low LNANAlycopene cyclaser; Phytoene SynthaselowNAlowphytoene synthasehp-1; high-pigment-1**highhighNANot cloned (lightsignaling)Nr-2: Never-ripe-2lowNANANot clonedGr: Green-ripelowNANANot clonedt; tangerinelowNANANot clonedat; apricotlowNANANot clonedCnr; Clear non-ripeninglowNANANot clonedL. esculentum ×low-highlow-highlow-highL. pennelliiRecombinant InbredsACO; ACC oxidase*lowNANAethylene BiosynthesisACS; ACC synthase*lowNANAethylene BiosynthesisACD; ACC deaminase*lowNANAethylene BiosynthesisTCTR1; tomato CTR1*low-highNANAethylene signalingMAPKKKThe dashed line separates mutants for which the corresponding gene has been cloned (1st tier) from those which have not (2nd tier). The last tier indicates transgenic lines altered in ethylene synthesis or response and with corresponding changes in carotenoid accumulation. Genotypes indicated with an (*) represent those for which multiple independent transgenic lines are available demonstrating a range of carotenoid accumulation levels.**Three different mutant alleles of hp-1 each having varying degrees of effect on carotenoid and flavonoid accumulation were provided by M. Koornneef.B = β-carotene.L = lycopene.While quantitative data for vitamin C and volatiles are unknown for many of these lines (NA), their respective phenotypes suggest they are likely to be altered in one or both.
In the case of flavor volatiles, the pathways for synthesis are in many cases not well established. For example, synthesis of apocarotenoids such as β-ionone and β-damascenone is not at all understood. Only recently has an Arabidopsis enzyme, CCD1 (Carotenoid Cleavage Dioxygenase), that synthesizes apocarotenoids such as β-ionone in vitro been identified (Schwartz et al., 2001). This gene is part of a multigene family, some of which are responsible for synthesis of other apocarotenoids such as ABA (Tan et al., 1997). CCD 1 cleaves multiple carotenoid substrates at the 9-10 and 9′-10′ bonds, potentially releasing volatiles such as β-ionone, although this has not been established in vivo. Similarly, several different volatiles are derived from lipid breakdown (Table 2). The likely first step in their syntheses is the action of a lipoxygenase (LOX) (Riley and Thompson, 1997; Baldwin et al., 2000). Currently there are 14 different EST contigs in the tomato database putatively identified as LOX. Any LOX exhibiting correlation with the lipid-derived volatiles would be a candidate sequence for analyses. It is exactly this sort of correlative biochemical and expression approach that resulted in identification of a key enzyme in strawberry volatile synthesis (Aharoni et al., 2000).
TABLE 2The 16 most significant flavor volatiles of tomatoConc.Log odorOdorVolatile(ppb)unitsPrecursorCharacteristicscis-3-Hexenal12,0003.7lipidTomato/greenβ-ionone42.8carotenoidfruity/floralHexanal3,1002.8lipidgreen/grassyβ-Damascenone12.7carotenoidFruity1-Penten-3-one5202.7lipidfruityfloral/green2 + 3-Methylbutanal272.1ILE/LEUMustytrans-2-Hexenal2701.2lipidGreen2-Isobutylthiazole361.0LEUTomato vine1-nitro-2-Phenylethane170.9PHEmusty, earthytrans-2-Heptenal600.7lipidGreenPhenylacetaldehyde150.6PHEfloral/alcohol6-Methyl-5-hepten-1300.4carotenoidfruity, floral2-onecis-3-Hexenol1500.3lipidGreen2-Phenylethanol1,9000.3PHENutty3-Methylbutanol3800.2LEUearthy, mustyMethyl salicylate480.08PHEwintergreenVolatiles are ranked by importance based on Odor Units (concentration X humans' ability to detect). Concentrations are average values from typical commercial tomatoes. Odor characteristics were determined by a trained expert panel.